The present invention is in the field of biological separations and processes.
In a variety of liquids containing cellular material, such as those used in fermentation processes, it is often desirable to be able to efficiently separate cellular products, such as cell bodies, lysed cells, and their breakdown products. It is often desirable to be able to remove or separate cellular products even in instances involving viscous liquids.
Accordingly, it is a general object of the present invention to be able to have available, a process for efficiently and completely removing or separating cellular products from liquid media, even in instances involving viscous liquids.
The process of the present invention can be applied inter alia to fermentation processes. Current industrial fermentations in conventional stirred tank fermentors for production of xanthan gum and other polysaccharides are energy-intensive and costly, mainly because the high broth viscosity causes agitation and aeration to be difficult and limits the final product concentration and productivity.
The production of xanthan gum is described here as an example of some of the problems encountered in biological processes that are addressed by the present invention, although the invention is not limited to that application.
Xanthan gum is a microbial polysaccharide widely used as a suspending, stabilizing, or thickening agent in the food industry. It is also used as a lubricant, emulsifier, or mobility-control agent in the oil-drilling industry. Presently, commercial xanthan gum is produced from glucose or dextrose by batch fermentation with the bacterium Xanthomonas campestris; the produced xanthan gum is then recovered and partially purified using alcohol precipitation. The final product usually also contains some cells and cell debris; however, it is desirable to produce xanthan gum product that is free of any particulates or cells, particularly for applications in oil recovery. The production of cell-free xanthan broth also allows for efficient concentration of xanthan fermentation broth by ultrafiltration without significant membrane fouling caused by cells and their debris (e.g., DNA and RNA) that would otherwise be present in the xanthan broth.
The present industrial process for xanthan gum production is energy-intensive and costly, mainly because the highly viscous xanthan broth causes agitation and aeration to be difficult in conventional stirred tank fermentors. Consequently, conventional xanthan gum fermentation has low xanthan concentration (usually below 3% wt/v) and low productivity (usually below 0.5 g/Lxc3x97h). There have been many attempts to increase xanthan productivity and to lower energy costs by using new agitation designs, and new types of bioreactors. Fermentation with water-in-oil emulsion and cell immobilization using porous Celite beads, which reduces broth viscosity and improves aeration and oxygen transfer, have also been studied. Although a high xanthan concentration of xcx9c5% was achieved in these processes, separating and recovering xanthan gum from the oil emulsion or Celite particles, though feasible, was difficult.
There have been only a few studies of xanthan fermentation using immobilized cells. Robinson and Wang (1988) used porous Celite beads to immobilize cells in xanthan fermentation. It is not clear, however, if the xanthan broth so produced was free of cells. Furthermore, a large portion of the xanthan product was trapped in the beads and could not be easily separated from the cells. Lebrun et al. (1994) studied polysaccharide production by cells immobilized in composite agar layer/microporous membrane structures, but concluded that the immobilized-cell system was not appropriate for xanthan gum production. It is clear that cell entrapment is not an appropriate cell immobilization method for xanthan gum fermentation because of the high viscosity of xanthan solution. The viscosity of the xanthan solution is high even at a low concentration. In batch xanthan fermentation, the broth viscosity has been found to reach more than 3000 cp at 2% (wt/v) xanthan concentration. The high viscosity of xanthan broth presents a major challenge in separating cells and cell debris from broth at industrial scale using conventional separation techniques, such as microfiltration, flocculation, and centrifugation. Thus, one of the objects of the present invention is to find an economical way to produce cell-free xanthan broth by either cell immobilization during fermentation or cell removal after fermentation.
One of the objects of the present invention is to provide a fermentation method which allows for the efficient and substantially complete removal of cells and cell debris from fermentation broths, even in instances involving viscous fermentation broths.
It is also an object of the present invention to allow for the removal of cellular products from liquids used as media for cellular reactions, such as fermentation broths, even those that are unusually viscous.
It is also an object of the present invention to produce an apparatus for carrying out the separation/removal process and cellular reactions of the present invention.
In view of the present disclosure, other advantages and the solutions to related problems may become apparent to one of ordinary skill in the art.
The present invention includes a process for separating cells form a liquid media, a method of fermentation using such a separation, and an apparatus for conducting such a separation or for facilitating a cellular reaction.
In broadest terms, the separation process of the present invention is a process for separating cells from a liquid containing cells. The process comprises the steps of: (a) bringing the liquid containing the cells into contact with one or more microbial polysaccharide and a fibrous material so as to adsorb the cells onto the fibrous material; and (b) separating the liquid from the fibrous material so as to remove the cells from the liquid. This may be done either by having the microbial polysaccharide(s) be in the liquid or by having the fibrous material be pre-treated with the microbial polysaccharide(s). Either way, a polysaccharide-mediated adsorption of the cells onto the fibrous material is brought about.
The process of the present invention may be used to remove cells from any liquid, but such liquids typically will be aqueous solutions, such as growth media, biological fluids, diagnostic samples, aqueous test samples, etc.
As referred to with respect to the present invention, the term xe2x80x9ccellsxe2x80x9d shall be understood to include, without limitation, cellsxe2x80x94alive, dead or attenuatedxe2x80x94and cell portions such as lysed cell walls, cell bodies, organelles, chromosome material and mixtures thereof.
The microbial polysaccharide(s) may be of any type. Naturally, there are a wide variety of microbial polysaccharides, such as the more well characterized and named polysaccharides selected from the group consisting of xanthan gum, dextran, pullulan, the polysaccharide types they represent and mixtures thereof.
The liquid may be brought into contact with the microbial polysaccharide and a fibrous material through any appropriate means, such as through the use of tanks and vats, liquid flows, etc.
The fibrous material used in accordance with the present invention may be any natural or synthetic fiber, and may for instance be selected from the group consisting of looped cotton terry cloth, cotton fabric sheet cloth, 50% cotton-50% polyester fabric sheet cloth, and polyester fabric sheet cloth. It has been found that cotton, particularly looped cotton terry cloth and cotton fabric sheet cloth, works best, with looped cotton terry cloth being most preferred.
The fibrous material may be in any non-woven, woven or geometrical arrangement (e.g., sheets, rolls, strands, threads, etc.), generally referred to as the xe2x80x9cfibrous matrix,xe2x80x9d and naturally may be produced and arranged so as to afford efficient contact with the liquid.
The liquid may be brought into contact with the microbial polysaccharide(s) and the fibrous material through any arrangement. Such arrangements may include the use of a liquid container into which a matrix of the fibrous material is placed. As an alternative, the liquid may be brought into contact with the fibrous material by causing the liquid to flow through, within or over a fibrous matrix of the fibrous material. Either the liquid may be moved relative to the fibrous matrix, or vice versa, such as through a liquid flowing over or through the matrix or by having the fibrous matrix mounted on frames and swung or agitated within a container or within a flow of a liquid, etc. The liquid container may also be moved with respect to the liquid, such as through agitation or oscillation. In a preferred embodiment, especially for viscous liquids, the liquid may be brought into contact with the fibrous material by pumping or otherwise forcing the liquid through a fibrous matrix of the fibrous material. This may be done by passing the liquid through a rotating fibrous matrix of the fibrous material through application of centrifugal force, such as by placing the liquid in the interior of the fibrous matrix and spinning it so as to move the liquid from its interior to its outer surface, through application of centrifugal force.
The ultimate separation of the cells from the remaining liquid also may be done through any appropriate means, such as through draining the liquid from the fibrous matrix, the physical removal of the fibrous matrix from the liquid, etc., as each application may require.
The separation process of the present invention, as summarized above, may be used as part of a method for producing a product liquid containing one or more reaction product(s) of a cellular reaction, which liquid is substantially free of cells. In general terms, the method includes the steps: (a) preparing a reaction mixture of: (i) water; (ii) one or more substrate substance(s) for the cellular reaction; (iii) cells of an organism capable of converting the substrate substance(s) to one or more reaction product(s); and (iv) one or more microbial polysaccharide(s); and (b) allowing the reaction mixture to undergo the cellular reaction so as to form the at least one reaction product from the at least one substrate substance; followed by (c) bringing the reaction mixture into contact with a fibrous material so as to adsorb the cells onto the fibrous material, and (d) separating said fibrous material from the reaction mixture so as to remove the said cells from the reaction mixture.
As referred to with respect to the production methods of the present invention, it will be understood that xe2x80x9ccellular reactionxe2x80x9d is intended in its broadest sense and may be any chemical reaction and/or physical change brought about by or catalyzed by cells of an organism. Such cells may be those of one-celled organisms or the cells of multi-celled organisms, whether microbial plant or animal. Such cellular reaction may be those carried out for industrial production purposes, or for pure or applied research. Typically, cellular reactions in which the present invention may be applied will include those that rely upon enzymatic reactions, both anabolic and catabolic.
The cellular reactions may involve one or more substrate substances which are broadly intended to mean any one or more substances that are the subject of the cellular reaction. In turn, the cellular reaction may produce one or more reaction products. Such product(s) may be obtained through the use of reaction conditions appropriate to the cellular reaction of interest.
Once the reaction product(s) is/are formed, the reaction mixture is brought into contact with a fibrous material so as to adsorb the cells onto the fibrous material. The fibrous material may then be separated from the reaction mixture so as to remove the cells from the reaction mixture.
In a variation of the basic cellular reaction process, it may be the case that the substrate substance(s) and/or reaction product(s) comprise(s) one or more microbial polysaccharide(s) and thus supplies the microbial polysaccharide(s) required in the method of the present invention in the form of a substrate or reaction product. In such cases, the microbial polysaccharides(s) need not be added.
In another variation, the microbial polysaccharides may be applied to the fibrous material, rather than being put into the liquid or arising from the cellular reaction.
The present invention also includes a method for producing xanthan gum solution substantially free of cells using the separation process of the present invention as summarized above. The method generally comprises the steps: (a) preparing a fermentation broth mixture of: (i) water; (ii) a saccharide selected from the group consisting of glucose, dextrose and mixtures thereof; and (iii) Xanthomonas campestris bacterial cells; (b) allowing the fermentation broth mixture to undergo fermentation so as to form xanthan gum polysaccharide in the fermentation broth mixture; followed by (c) bringing the fermentation broth mixture into contact with a fibrous material so as to adsorb the bacterial cells onto the fibrous material, and (d) separating the fibrous material from the fermentation broth mixture so as to remove the cells from the fermentation broth mixture (herein xe2x80x9cseparatingxe2x80x9d is intended broadly, whether removing the fibrous material from the broth or allowing the broth to flow from the fibrous material).
The invention also includes an apparatus that may be used for carrying out the separation process of the present invention, and which also may be used in accordance with the production methods using the separation process. The apparatus in broad terms is one for separating cells from a liquid containing the cells and comprises: (a) a matrix of a fibrous material treated with one or more microbial polysaccharide(s) having an interior and an outer surface; (b) liquid application dispenser for dispensing the liquid into the interior of the matrix of the fibrous material; and (c) an apparatus to separate the liquid form the matrix of fibrous material. For example, a spinner may be adapted to spin the matrix of a fibrous material such that the liquid, once in the interior of the matrix of the fibrous material, moves out of the outer surface of the matrix of the fibrous material. The fibrous material may become treated with one or more microbial polysaccharide(s) through microbial polysaccharide(s) being resident in the liquid either naturally occurring or arising as a result of a cellular reaction. In an alternative embodiment to those described, where the fibrous material is not pretreated with microbial polysaccharide(s), the apparatus may contain a liquid to be supplied to the liquid application dispenser, the liquid containing at least one microbial polysaccharide and cells of an organism capable of converting the substrate substance(s) to the reaction product(s), such that when the liquid is brought into contact with the matrix of said fibrous material, said cells become adsorbed onto said fibrous material.
The apparatus may additionally include a recirculator adapted to recirculate liquid moving out of the outer surface of the mass of the fibrous material into the interior of the mass of the fibrous material, in order to increase the exposure of the polysaccharide-treated fibrous material to the cells over time. Such a device may be in the form of a recirculating pump or other appropriate flow control device.
The invention also includes an apparatus for facilitating a method for producing a cellular reaction product in a liquid, the apparatus comprising: (a) a matrix of a fibrous material having an interior and an outer surface, the fibrous material treated with at least one microbial polysaccharide and having adsorbed thereupon cells of an organism capable of converting the at least one substrate substance to the at least one reaction product; and (b) a liquid circulator device for circulating the liquid through the matrix of the fibrous material.
The invention also includes an apparatus for facilitating a method for producing a cellular reaction product in a liquid, the apparatus comprising: (a) a matrix of a fibrous material having an interior and an outer surface, the fibrous material treated with at least one microbial polysaccharide and having adsorbed thereupon cells of an organism capable of converting the at least one substrate substance to the at least one reaction product; (b) a reaction vessel for holding the liquid; (c) a liquid transport device adapted to move the matrix of the fibrous material relative to the liquid, such as through agitation or oscillation, for example.
The present invention also includes an apparatus for facilitating a method for producing a cellular reaction product in a liquid. The apparatus comprises a matrix of a fibrous material having an interior and an outer surface, the fibrous material treated with at least one microbial polysaccharide and having adsorbed onto it cells of an organism capable of converting one or more substrate substance to one or more reaction product. The apparatus also includes a liquid application dispenser for dispensing the liquid into the interior of the matrix of the fibrous material; and a spinner adapted to spin the matrix of a fibrous material such that the liquid, once in the interior of the matrix of the fibrous material, moves through the matrix of a fibrous material toward the outer surface of the matrix of the fibrous material so as to bring the liquid in contact with the cells of the organism.
In an alternative embodiment to those described above, the fibrous material is not pretreated with microbial polysaccharide(s), and in such case the apparatus may contain a liquid to be supplied to the liquid application dispenser, the liquid containing at least one microbial polysaccharide and cells of an organism capable of converting the substrate substance(s) to the reaction product(s), such that when the liquid is brought into contact with the matrix of the fibrous material, the cells become adsorbed onto the fibrous material.
The apparatus of the present invention in all its embodiments may include fluid containment and/or conduction apparatus to allow the operator to cause (1) the liquid to flow through a fibrous matrix of the fibrous material, (2) pump the liquid through a matrix of the fibrous material, (3) agitate the liquid in a vessel containing a matrix of the fibrous material; (4) pass a matrix of the fibrous material through the liquid, (5) agitate a matrix of the fibrous material within the liquid and/or (6) force the liquid through a rotating matrix of the fibrous material through application of centrifugal force. Such apparatus may be supplied using liquid containment and/or conduction devices known in the art and which could be applied to bring about the desired result in accordance with the parameters of the specific separation or reaction to which the apparatus is to be applied. Examples may include arrangements of reaction vats, pumps and liquid conduits to contain the liquid and bring it into contact with the fibrous matrix.
The apparatus may additionally include a recirculator adapted to recirculate liquid within the fibrous matrix, such as by moving out of the outer surface of the mass of the fibrous material into the interior of the mass of the fibrous material so as to increase the exposure of substrate to the adsorbed cells over time.
In applications involving viscous liquids, it is preferred that the fibrous matrix be exposed to air and spun at such a rate that such that the viscosity is overcome by the mechanical shearing.
The high viscosity of a xanthan solution at low concentration presents a major challenge in agitating xanthan broth during fermentation. Xanthan solution, however, shows a high degree of pseudoplasticity, i.e., the viscosity decreases rapidly as the shear rate increases. This shear-thinning property allows efficient pumping of xanthan polymer at high pumping (shear) rates. The present invention thus includes a centrifugal, fibrous-bed bioreactor that may be used for instance for viscous xanthan gum fermentation, and in other processes that present similar problems of viscosity and the need for contact with cells. Difficulties in agitation and aeration in the conventional stirred-tank bioreactors, such as those used in traditional fermentation, are overcome by continuous medium recirculation through a rotating fibrous matrix, which contained immobilized cells. In this bioreactor, liquid media and air (or other oxygen-containing gas) were passed through the porous fibrous matrix to ensure intimate contact with the immobilized cells, thus achieving high oxygen transfer and reaction rates. The centrifugal force generated from rotating the fibrous matrix separated the xanthan polymer from the immobilized cells, thus producing a cell-free xanthan broth. It is important to produce xanthan gum solution free of any particulates or cells for applications in oil recovery. The production of cell-free xanthan broth also allows one to efficiently concentrate the xanthan fermentation broth by ultrafiltration without significant membrane fouling caused by cells and resulting debris (e.g., DNA and RNA) that would otherwise be present in the xanthan broth.